Cell detection is a fundamental procedure in any biomedical study where microscopy images of cell populations are used. Cell detection can be used for counting the individual cells, or as a basis for further analysis, ranging from feature extraction to single cell tracking. This procedure has been intensively studied in the image processing community.
Fluorescence microscopy is the standard tool for detection and analysis of cellular phenomena. This technique, however, has a number of drawbacks such as the limited number of available fluorescent channels in microscopes, overlapping excitation and emission spectra of the stains, and phototoxicity.
The development of highly specific stains and probes, for example the green fluorescent protein and its derivatives, have made fluorescence microscopy the standard tool for visualization and analysis of cellular functions and phenomena. On the other hand, automated microscopes and advances in digital image analysis have enabled high-throughput studies automating the imaging procedure and cell based measurements. In fluorescence microscopy of eukaryotic cells, automated single-cell quantification can be achieved using multiple fluorescent probes and channels in a single experiment. The first fluorescence channel enables detection of stained nuclei, resulting in markers for cell locations. The second fluorescent channel visualizes the areas occupied by whole cells or cytoplasm, for example by a cytoskeletal actin stain, as described in Moffat J, Grueneberg D A, Yang X, Kim S Y, Kloepfer A M, et al. (2006) A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124: 1283-1298. Alternatively, a nonspecific subcellular stain can be used for whole cell detection. Regardless of the approach for whole cell staining, cells that are touching or partly overlapping can be automatically separated with the help of the nuclei markers of the first channel, as describe in Carpenter A E, Jones T R, Lamprecht M R, Clarke C, Kang I H, et al. (2006) CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol 7: R100. Finally, subcellular phenomena are quantified by measuring different properties of the first and second channels, or by using additional organelle and molecule specific probes and extra fluorescence channels, for example in colocalization measurements, as described in Bolte S, Cordelières F P (2006) A guided tour into subcellular colocalization analysis in light microscopy. J Microsc 224: 213-232.
Because of the limited number of fluorescent channels available, and because of partly overlapping excitation and emission spectra of the probes, studies involving subcellular colocalization are commonly carried out without nuclear or whole cell staining. As a consequence, cell-by-cell measurements are not possible. Single cell measurements are also difficult or even impossible in cells that are used for negative control, where the lack of fluorescence is used for the detection of some phenomena. Furthermore, there are other limitations in fluorescence microscopy, such as phototoxicity and imaging setup complexity. These problems have motivated the search for alternate methods to replace at least some of the fluorescence channels with standard transmitted light microscopy.
A number of problems in counting cells using the conventional methods have been observed. These problems include the contamination of the cells (and possibly the growth culture in which they are found) with extraneous chemical substances that are needed for counting, but that make the further use of the cells impractical or impossible.
There is a need for systems and methods that provide the ability to count cells without subjecting the cells to extraneous chemicals.